Synthesis of methoxy- and bromo-substituted indirubins and their activities on apoptosis induction in human neuroblastoma cells

Article abstract of DOI:10.1016/j.bmcl.2011.07.011

This paper reports the synthesis of methoxy- and bromo-indirubins, and their antiproliferative activities in human neuroblastoma. Among 20 compounds, 5'-methoxyindirubin induced cell death in human neuroblastoma cells (IMR-32, SK-N-SH and NB-39) without inhibiting normal cells (NHDF and HUVEC). Typical morphologic features of apoptosis were observed in 5′-methoxyindirubin- treated cells by Hoechst 33342 staining. Additional studies by flow cytometry support apoptosis induction. These data suggest that 5′-methoxyindirubin might be an effective drug for treatment of neuroblastoma.

Full text of DOI:10.1016/j.bmcl.2011.07.011

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5370–5373
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of methoxy- and bromo-substituted indirubins and their activities
on apoptosis induction in human neuroblastoma cells
Hiroaki Saito a, Keiichi Tabata a, Satoshi Hanada a, Yuko Kanda a, Takashi Suzuki a,b, Shinichi Miyairi a,
⇑
a School of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi-shi, Chiba 274-8555, Japan
b School of Medicine, Nihon University, 30-1 Oyaguchikami-cho, Itabashi-ku, Tokyo 173-0032, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 March 2011
Revised 2 July 2011
Accepted 6 July 2011
Available online 14 July 2011
This paper reports the synthesis of methoxy- and bromo-indirubins, and their antiproliferative activities
in human neuroblastoma. Among 20 compounds, 50-methoxyindirubin induced cell death in human
neuroblastoma cells (IMR-32, SK-N-SH and NB-39) without inhibiting normal cells (NHDF and HUVEC).
Typical morphologic features of apoptosis were observed in 50-methoxyindirubin-treated cells by Hoe-
chst 33342 staining. Additional studies by flow cytometry support apoptosis induction. These data
suggest that 50-methoxyindirubin might be an effective drug for treatment of neuroblastoma.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Indirubin
Methoxyindirubin
Neuroblastoma
Apoptosis
Tumor selectivity
Neuroblastoma is the most common extracranial solid tumor in
children and is derived from cells of the sympathetic nervous sys-
tem.1,2 Approximately 40% of patients with neuroblastoma are
infants, and the incidence decreases with age. Most patients
diagnosed with neuroblastoma are less than 10 years old. Its prog-
nosis varies according to patient age at diagnosis. In patients more
than 1 year old, the tumor is very aggressive and drug-resistant.3
The 5-year survival rate of patients with such advanced tumors
is only 25ꢀ30%, despite aggressive chemotherapy.4 However, some
pseudo-benign neuroblastomas, especially occurring in infants and
children less than 1 year old, are known to regress spontaneously
or to mature even if metastases to bone marrow, skin and/or liver
(stage IVs) are present. Mechanisms of spontaneous regression still
remain unclear, but delayed apoptosis or differentiation are
suggested.5 It is well established that apoptosis is an important
mechanism in the normal development of nervous systems.
However, neuroblastoma is derived from neural crest cells when
apoptotic systems do not function properly. It is further suggested
that resistance to apoptosis plays a contributory role in the mech-
anism of aggressive behavior shown by advanced neuroblastoma.6
Accordingly, compounds exhibiting apoptosis-inducing activity on
neuroblastoma cells specifically may contribute to a cure for
advanced neuroblastoma.
CAKI-2 (renal cell cancer),7 HeLa cell (cervical cancer),8 HepG2 cell
(hepatoma),8 HCT116 cell (colon cancer),8 and Hep-2 cell (laryn-
geal carcinoma).9 Induction of caspase-independent cell death,
possibly via necrosis, by its 7-bromo derivative has also been re-
ported.10 Since available indirubin derivatives have been limited,
systematic screening to find lead compounds for novel cancer
chemotherapy drugs has also been limited. In the present study,
we aimed to identify compounds with potential to induce apopto-
sis in neuroblastoma cells, but not in normal cells, by systematic
screening.
For systematic screening, we prepared indirubins with two dif-
ferent types of substituents on the aromatic rings (Fig. 1). One was
a methoxy group, a typical electron-donating group, and another
was bromine, a typical electron-withdrawing group. Some of these
compounds were also converted into 30-oxime derivatives (14–20)
by reaction with hydroxylamine in pyridine.11 The indirubins with
a substituent at R2–4 (2–4, 9–11) and 50-bromoindirubin (12) were
prepared by the usual manner: condensation of the corresponding
isatins with indoxyl acetate (5-bromoindoxyl acetate for 12) in the
presence of Na2CO3 in methanol.11 In contrast, indirubins with a
methoxy group at 50- or 60-position (5 and 6) and bromine at 60-po-
sition (13) were synthesized by the reaction between oxindole and
2-chloroindol-3-one derivatives prepared from the corresponding
isatins (Scheme 1).12–14
Recently apoptosis-inducing activity of indirubin 30-oxime has
been revealed with human cell lines such as A498, CAKI-1 and
We examined the effect of indirubins on cell viability with three
human neuroblastoma cell lines (group I), IMR-32,15–18 SK-N-SH18
and NB-3916–18, and with two normal cell lines (group II), NHDF
(normal human dermal fibroblast)19,20 and HUVEC (human
⇑
Corresponding author. Tel.: +81 47 465 6290; fax: +81 47 465 6353.
E-mail address: miyairi.shinichi@nihon-u.ac.jp (S. Miyairi).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.07.011

H. Saito et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5370–5373
5371
1
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concentration (logM)
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-7
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concentration (logM)
concentration (logM)
5
17
Figure 1. Structures of indirubins (1ꢀ20).
120
100
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40
20
0
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0
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concentration (logM)
concentration (logM)
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Scheme 1.
0
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concentration (logM)
concentration (logM)
19
7
umbilical vein endothelial cell)21,22 (Fig. 2). Our criteria for screen-
ing the potential of a compound as an anti-neuroblastoma drug is
that cell viability of the cells in group I decreases to less than 50%
while that of the cells in group II remain more than 90% at certain
concentrations. Indirubin (1) and its 6- or 7-methoxylated
derivatives (3 and 4) did not show any significant effect on cell via-
bility of cells in both groups at the concentrations examined (up to
120
100
80
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0
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0
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concentration (logM)
concentration (logM)
8
40 lM). 5-Methoxyindirubin (2) was cytotoxic, but no significant
120
100
80
60
40
20
0
IMR-32
SK-N-SH
NB-39
NHDF
HUVEC
differences were observed between the cells in groups I and II. In
contrast to the indirubins having methoxy group at R2–4, 50- and
60-methoxyindirubins (5, 6) exhibited selective toxicity to cells in
group I. Although 50-methoxyindirubin (5) satisfied the criteria
described above, the 60-isomer (6) had significant cytotoxicity to
Group I
Group II
0
-7
-6
-5
-4
concentration (logM)
the cells in group II at 40 lM (82% in HNDF; 60% in HUVEC).
To determine the effectiveness of the methoxy moiety at
position 50, we prepared 50-alkylindirubins as methoxy analogs.
Interestingly, 50-methylindirubin (7) and 50-ethylindirubin (8)
showed very weak cytotoxicities in both groups I and II up to
Figure 2. Cytotoxicity of indirubins on neuroblastoma cells and normal cells. Cell
viability was measured using an MTT assay. IMR-32 (filled circles), SK-N-SH (filled
squares), NB-39 (filled diamonds), NHDF (open circles), and HUVEC (open squares)
cells were treated with the indicated concentrations of indirubins or DMSO (vehicle
control) for 48 h. Each plot shows the survival rate relative to the vehicle control
[mean standard error of the mean (SEM), n = 3].
40
l
M. Indirubins with bromine at R5,6 (12 and 13) as well as
R2–4 (9–11) did not show any significant effects on cell viability

5372
H. Saito et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5370–5373
in either group (50-bromoindirubin (12) is depicted as an example).
Kim et al. recently mentioned an important effect between the
bromine atom at position 50 and cyclin-dependent kinase 2
(CDK2) based on a docking model simulation.23 The electrostatic
interaction between the lone pair electrons of the 50-bromine
group and the positively charged Lys89 residue may result in a
higher binding affinity with CDK2. In our study, comparing 50-
methoxyindirubin (5) with 50-alkylindirubins (7, 8), much stronger
cytotoxicities and selectivities against neuroblastoma were
observed in 50-methoxy analogs (5). These preferable effects might
result from additional electrostatic effects of lone pair electrons
and positively charged amino acids. However, bromine substitu-
tion at position 50 does not exhibit positive results in this case.
We then focused on 30-oxime derivatives because apoptosis-
inducing potential of indirubin 30-oxime (14) has been found in
human cells.7–9 Since this derivatization makes indirubin more
soluble, we expected biological availability and efficacy to increase.
All of the compounds gained additional cytotoxicity by this deriv-
atization as presumed. Unexpectedly, 50-methoxyindirubin (5) lost
the selective cytotoxicity by conversion to 30-oxime (18). On the
other hand, indirubin 30-oxime (14) and its 6-methoxylated deriv-
ative (16) gained cytotoxicity selective to the cells in group I, while
the other 30-oxime derivatives (15, 17) including bromoindirubins
(6-bromoindirubin 30-oxime (19) is depicted as an example)
exhibited cytotoxicity non-specifically. However, indirubin 30-
oxime (14) exhibited weak cytotoxicity to group II cells, too.
6-Methoxyindirubin 30-oxime (16) was not cytotoxic to group II
cells, but the cytotoxicity was not sufficient to some cells in group
I. Accordingly, we chose 50-methoxyindirubin (5) for further study.
To determine participation of apoptosis on cytotoxicity induced
by 50-methoxyindirubin (5), we subjected IMR-32 cells to two
Figure 3. Typical morphologic features of apoptosis revealed using Hoechst 33342
staining.IMR-32cellswereexposedto5(1 ꢁ 10ꢀ6–1 ꢁ 10ꢀ4 M)orCDDP(1 ꢁ 10ꢀ4 M)
for 24 h and phase-contrast images (upper), and fluorescence images (lower) were
obtained.
Figure 4. Analysisofapoptosisusingflowcytometry.(A)IMR-32cellsweretreatedwith5(1 ꢁ 10ꢀ4)orDMSO(asavehiclecontrol(V))for48 h.Normalcells(N)weretheuntreated
group.TheverticalaxisindicatesPI(FL4 Log)andthehorizontalaxisindicatesAnnexinV-AlexaFluor488(FL1 Log). B. Percentagesofcellpopulationsineacharea(Earlyapoptosis:
rightlower,Lateapoptosis:rightupper).⁄p <0.05,⁄⁄p <0.01versusvehiclecontrolascomparedusingaone-wayanalysisofvariance(ANOVA)followedbyBonferroni’sposthoctest.

H. Saito et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5370–5373
5373
different experiments. After 24-h incubation of the cells in the
presence or absence of compound 5, the cells were stained with
Hoechst 33342, and morphological changes were observed by fluo-
rescence microscopy (Fig. 3).24 In the control experiment, the nu-
clei of the neuroblastoma cells were round in shape and stained
homogenously. The cells treated with 50-methoxyindirubin (5)
showed typical morphological features of apoptosis such as cell
shrinkage, chromatin condensation and fragmented fluorescent
nuclei like that of authentic apoptosis inducer, cisplatin (CDDP).25
Moreover, cells were subjected to flow cytometry after Annexin V
and propidium iodide (PI) double staining.26 Figure 4A shows the
distribution of stained cells prepared from vehicle control and 50-
Nihon University) for performing the mass measurement. This re-
search was supported in part by a grant from a Nihon University
Multidisciplinary Research Grant for S.M. (2010ꢀ2011).
References and notes
1. Bolande, R. P. Hum. Pathol. 1974, 5, 409.
2. Nakagawara, A.; Ohira, M. Cancer Lett. 2004, 204, 213.
3. Torkin, R.; Lavoie, J. F.; Kaplan, D. R.; Yeger, H. Mol. Cancer Ther. 2005, 4, 1.
4. Tonini, G. P.; Pistoia, V. Curr. Pharm. Design 2006, 12, 2303.
5. Maris, J. M.; Matthay, K. K. J. Clin. Oncol. 1999, 17, 2264.
6. Poulaki, V.; Mitsiades, N.; Romero, M. E.; Tsokos, M. Cancer Res. 2001, 61, 4864.
7. Perabo, F. G. E.; Landwehrs, G.; Frössler, C.; Schmidt, D. H.; Mueller, S. C. Urol.
Oncol-Semin. Ori. 2009, in press.
8. Shi, J.; Shen, H.-M. Biochem. Pharmacol. 2008, 75, 1729.
9. Kameswaran, T. R.; Ramanibai, R. Biomed. Pharmacother. 2009, 63, 146.
10. Ribas, J.; Bettayeb, K.; Ferandin, Y.; Knockaert, M.; Garrofé-Ochoa, X.; Totzke, F.;
Schächtele, C.; Mester, J.; Polychronopoulos, P.; Magiatis, P.; Skaltsounis, A.-L.;
Boix, J.; Meijer, L. Oncogene 2006, 25, 6304.
11. Hoessel, R.; Leclerc, S.; Endicott, J. A.; Nobel, M. E. M.; Lawrie, A.; Tunnah, P.;
Leost, M.; Damiens, E.; Marie, D.; Marko, D.; Niederberger, E.; Tang, W.;
Eisenbrand, G.; Meijer, L. Nat. Cell. Biol. 1999, 1, 60.
12. Katritzky, A. R.; Fan, W.-Q.; Koziol, A. E.; Palenik, G. J. J. Heterocycl. Chem. 1989,
26, 821.
methoxyindirubin (5) (100
of apoptotic cells were estimated based on Figure 4A, and dramat-
ically increased at 100 M concentration of the indirubin 5 in both
l
M) as examples.27 The percentages
l
early and late apoptosis (Fig. 4B). These results lead us to conclude
that the mechanism of cell death induced by 50-methoxyindirubin
(5) is apoptosis. Indirubins are known to inhibit cyclin-dependent
kinases (CDKs) by binding to their ATP-binding site.11 The inhibi-
tion of CDKs by indirubins arrests the cell cycle in the G1 and/or
G2/M phases, prohibits cell growth, and induces apoptosis.8,28–32
Since apoptotic mechanisms are involved in chemotherapeutic
sensitivity and spontaneous regression of neuroblastoma, 50-meth-
oxyindirubin (5) may have comparatively high potential against
neuroblastoma.
13. Bergman, J.; Lindström, J.; Tilstam, U. Tetrahedron 1985, 41, 2879.
14. Grimshaw, J.; Begley, W. Synthesis 1974, 496.
15. Tabata, K.; Motani, K.; Takayanagi, N.; Nishimura, R.; Asami, S.; Kimura, Y.;
Ukiya, M.; Hasegawa, D.; Akihisa, T.; Suzuki, T. Biol. Pharm. Bull. 2005, 28, 1404.
16. Nishimura, R.; Tabata, K.; Arakawa, M.; Ito, Y.; Kimura, Y.; Akihisa, T.; Nagai, H.;
Sakuma, A.; Kohno, H.; Suzuki, T. Biol. Pharm. Bull. 2007, 30, 1878.
17. Yamaguchi, Y.; Tabata, K.; Asami, S.; Miyake, M.; Suzuki, T. Biol. Pharm. Bull.
2007, 30, 638.
In summary, we prepared indirubins with two different types of
substituents, methoxy group or bromine, on the aromatic rings for
systematic screening. We found that 50- and 60-methoxy-indiru-
bins (5 and 6) showed significant cytotoxicity to neuro-blastoma
cells. When the carbonyl group at 30-position was oxime, cytotox-
icity appeared in every indirubin examined. In this case, indirubin
30-oxime (14) and its 6-methoxylated derivative (16) showed
selective cytotoxicity to neuroblastoma, while its 50-methoxylated
derivative (18) did not show any selectivity. Thus, it has been con-
cluded that 50-methoxyindirubin (5) is the most favorable indiru-
bin examined for specific induction of cell death in
neuroblastoma. Moreover, we concluded that the methoxy group
is preferable to induce selective cytotoxicity against neuroblas-
toma rather than alkyl or bromine (5 vs. 7, 8, and 12). It was further
clarified that 50-methoxyindirubin (5) induced early and late apop-
tosis in neuroblastoma cells. Further studies are needed to eluci-
date the mechanism of apoptosis by biochemical approaches.
Nonetheless, present data reinforce the anticancer potential of
indirubins.
18. Motani, K.; Tabata, K.; Kimura, Y.; Okano, S.; Shibata, Y.; Abiko, Y.; Nagai, H.;
Akihisa, T.; Suzuki, T. Biol. Pharm. Bull. 2008, 31, 618.
19. Postiglione, L.; Di Domenico, G.; Caraglia, M.; Marra, M.; Giuberti, G.; Del
Vecchio, L.; Montagnani, S.; Macri, M.; Bruno, E. M.; Abbruzzese, A.; Rossi, G.
Int. J. Oncol. 2005, 26, 1193.
20. Yanamoto, S.; Iwamoto, T.; Kawasaki, G.; Yoshitomi, I.; Baba, N.; Mizuno, A.
Cancer Lett. 2005, 223, 67.
21. Kimura, Y.; Taniguchi, M.; Baba, K. Plant. Med. 2004, 70, 211.
22. Kimura, Y.; Baba, K. Int. J. Cancer 2003, 106, 429.
23. Choi, S. J.; Lee, J. E.; Jeong, S. Y.; Im, I.; Lee, S. D.; Lee, E. J.; Lee, S. K.; Kwon, S. M.;
Ahn, S. G.; Yoon, J. H.; Han, S. Y.; Kim, J. I.; Kim, Y. C. J. Med. Chem. 2010, 53,
3696.
24. Sandhu, L. C.; Warters, R. L.; Dethlefsen, L. A. Cytometry 1985, 6, 191.
25. Piacentini, M.; Fesus, L.; Melino, G. FEBS Lett. 1993, 320, 150.
26. Pfaffel-Schubart, G.; Scalfi-Happ, C.; Rück, A. Med. Laser Appl. 2008, 23, 25.
27. We additionally determined cell survival of neuroblastoma and normal cells at
100 lM of compound 5. The survival rates of all three of the neuroblastoma cell
lines were approximately 10% while that of NHDF was more than 80%.
Unfortunately, HUVEC cells were damaged at this concentration and the
survival rate was approximately 40%.
28. Libnow, S.; Methling, K.; Hein, M.; Michalik, D.; Harms, M.; Wende, K.;
Flemming, A.; Köckerling, M.; Reinke, H.; Bednarski, P. J.; Lalk, M.; Langer, P.
Bioorg. Med. Chem. 2008, 16, 5570.
29. Nam, S.; Buettner, R.; Turkson, J.; Kim, D.; Cheng, J. Q.; Muehlbeyer, S.; Hippe,
F.; Vatter, S.; Merz, K. H.; Eisenbrand, G.; Jove, R. Proc. Natl. Acad. Sci. U.S.A.
2005, 102, 5998.
Acknowledgments
30. Kim, S.-A.; Kim, S.-W.; Chang, S.; Yoon, J.-H.; Ahn, S.-G. Cancer Lett. 2009, 274,
72.
31. Kim, S.-A.; Kim, Y.-C.; Kim, S.-W.; Lee, S.-H.; Min, J.-J.; Ahn, S.-G.; Yoon, J.-H.
Clin. Cancer Res. 2007, 13, 253.
32. Lee, J.-W.; Moon, M. J.; Min, H.-Y.; Chung, H.-J.; Park, E.-J.; Park, H. J.; Hong, J.-
Y.; Kim, Y.-C.; Lee, S. K. Bioorg. Med. Chem. Lett. 2005, 15, 3948.
The authors are grateful to Ms. Konomi Tanaka and Ms. Fumina
Amemiya for technical assistance in the experimental work. The
authors thank Dr. Toshimitsu Suzuki, Fukushima Medical Univer-
sity School of Medicine, for providing NB-39 cells. The authors also
thank Dr. Koichi Metori (Analytical Center, School of Pharmacy,